Tumors grow through two primary processes: proliferation and invasion. Proliferative growth represents the increase in size of the central tumor mass through the division of cells. Invasive growth occurs in tissues in the regions adjacent to and around the central tumor mass. In the invasion process, individual tumor cells detach from the central tumor mass and begin to actively move through the surrounding, non-tumorous tissue, either by compression or enzymatic degradation. The presumably highly branched cells formed by these invading cells represent a dynamically evolving network pattern. The chains formed in the invasive network can be as thin as a single cell in width. Further, the invading cells are significantly elongated along the direction in which they are traveling.
Malignant tumors such as highly malignant brain tumors (e.g., gliomas and glioblastoma multiforme) have several features such as proliferation, invasion, central necrosis/apoptosis and neo-vascularization. Tumors outside the central nervous system show extensive metastasis by way of the blood circulatory and lymphatic systems.
Several in vitro assays have been described that are designed to measure either cell proliferation, migration, or invasion. For example, a cell colony/spheroid-agarose assay uses cell suspensions within, or multicellular tumor spheroids placed on top of agarose (in a cell culture dish), which is then covered with cell culture medium. Carlsson J., Int. J. Cancer, 20:129-136, 1977. The assay is designed to study the growth dynamics of the cell colonies or spheroid. The medium superlayer distributes growth factors and growth limiting factors produced by the tumor, without preserving their important regional concentration differences (i.e., higher concentration around the tumor). Also, the superlayer has to be changed routinely, altering the environmental setting.
A 2D migration assay can be used to describe the movement of a cell population from a central area in an expanding circle. Giese A., Neurosurgery, 37:294-302, 1995. The cells are placed in a cell culture dish and covered with medium. Aside from the edge of the dish, there is no mechanical confinement and no chemo-gradients can be established due to “equalizing” in the medium superlayer.
Invasiveness assays (e.g., commercially available through Costar® as the 24-well Transwell™ System) use a medium-supplemented, two-chamber system, which is designed to detect cell migration between the two chambers. Repesh L. A., Invasion Metastasis, 9:192-208, 1989. The insert (top chamber) has a polycarbonate membrane on the bottom, which has a number of pores (8 μm size). After a certain number of cells are placed onto this membrane, they start to move through the pores and drop into the lower chamber where they either start anchorage-independent growth or eventually attach. After an observation period the cell number in both chambers is counted (Coulter Counter System®) and a ratio indicates the specific invasive potential of the cell line used. A variation of the assay uses a Matrigel® layer on top of the polycarbonate membrane to investigate the enzymatic activity of the cells to digest their way towards the pores.
A spheroid-fetal rat brain aggregate assay uses rat brain aggregates co-cultured on a medium/agar-layer and covered with a cell-culture medium that is changed routinely. Khoshyomn S., J. Neuro-Oncology, 38:1-10, 1998. The migration capacity of the tumor cells is determined by the destruction of the rat brain aggregate, not by the direct measurement of cell branches.